The invention is in the fields of recombinant cell lines, recombinant animal viral vectors, defective adenovirus vectors, subunit vaccines and gene therapy.
Adenoviruses have recently begun to be used as vectors for gene expression, recombinant subunit vaccines and gene therapy. Yeh et al. (1997) FASEB J. 11:615-623; and Imler (1998) Vaccine 13:1143-1151. They have been detected in many animal species, exhibit minimal pathogenicity, and are non-integrative. Adenoviruses are capable of infecting a wide variety of cell types, both dividing and quiescent, and have a natural tropism for airway epithelial cells. The advantages to the use of adenoviruses as vectors include suitability for genetic manipulation, ability to replicate to high titers, stability and ease of production. Adenoviruses have been used as live enteric viral vaccines for many years with an excellent safety profile.
Adenoviruses are distinguished according to the species of animal which they infect (e.g., human, bovine, canine, etc.). Particular species of adenoviruses are further characterized serologically, according to type.
The adenovirus E1 region encodes several functions that are essential to viral replication. The E1A region is responsible for encoding functions that activate early and late transcription, stimulate progression of infected cells into the S phase of the cell cycle, and antagonize the effects of xcex1- and xcex2-interferons. The E1B region encodes functions involved in stimulating cell-cycle progression of infected cells, blocking apoptosis in infected cells, and blocking nucleocytoplasmic transport of host cell mRNA. In addition, part or all of the E1 region is responsible for cell transformation. See, for example, Shenk, Adenoviridae: The viruses and their replication. In xe2x80x9cVirologyxe2x80x9d (B. Fields, ed.) Chapter 67, Lippincott-Raven, Philadelphia, 1996, pp. 2111-2148.
Ideally (for safety considerations), one or more essential regions of the adenovirus genome are inactivated in the genome of an adenoviral vector. For example, the E1 region, encoding several essential functions (see above) as well as potential adenovirus transforming functions, will be inactivated in many types of adenovirus vector. However, since the E1 regions are essential for normal virus replication, propagation of adenovirus vectors lacking all or part of the E1 regions requires a helper cell line that provides E1 functions. Heretofore, such helper cell lines have provided E1 function by including E1 sequences from the same adenovirus type that is propagated in the cell line. For example, the human 293 cell line, containing human adenovirus type 5 E1 sequences, can be used for the propagation of human adenovirus with a mutated E1 region. Graham et al. (1977) J. Gen. Virol. 36:59-72. Similarly, cell lines suitable for the propagation of E2- and E4-mutant adenoviruses have been described. Klessig et al. (1984) Mol. Cell. Biol. 4:1354-1362; Weinberg et al. (1983) Proc. Natl. Acad. Sci. USA 80:5383-5386.
Homologous recombination occurs readily between adenoviruses of the same type, both in the wild and during coinfection of cultured cells. Ginsberg et al. The Genetics of Adenoviruses. In: Fraenkel-Conrat H and Wagner R R eds., xe2x80x9cComprehensive Virologyxe2x80x9d volume 9, New York, Plenum Press, 1977; Takemori (1972) Virology 47:157-167; and Williams et al. (1975) Cell 4:113-119. Consequently, when a mutant adenovirus is passaged through a helper cell line containing homologous adenovirus sequences, homologous recombination can result in the generation of wild-type adenoviruses. For example, when replication-defective adenoviruses containing E1 deletions were passaged in a complementing cell line containing adenovirus E1 sequences, replication-competent viruses emerged, in which the deleted E1 region had been restored through recombination with homologous E1 sequences present in the helper cell. See, for example, Hehir et al. (1996) J. Virol. 70:8459-8467; Fallaux et al. (1998) Human Gene Therapy 9:1909-1917.
Accordingly, there is a need for an adenovirus vector-helper cell system in which vectors deleted for E1 can be propagated in a cell line providing E1 function, without the likelihood that wild-type virus will be generated by recombination between the vector genome and viral sequences in the helper cell.
It is an object of the invention to provide a system for the growth and propagation of replication-defective adenovirus vectors, wherein the system does not have the potential to produce recombinant, replication-competent adenoviruses.
Accordingly, the invention provides host cells, preferably bovine, that are permissive for the replication of a defective adenovirus vector, in particular, a recombinant adenovirus that is mutated in the E1 region of the adenovirus genome (i. e., the E1A region, the E1B region or both). The E1 mutation can be a deletion, insertion, substitution, one or more point mutation(s), a rearrangement, or any other type of in vivo or in vitro genetic change. Such defective adenovirus vectors will often comprise heterologous sequences. In adenovirus genomes with deletions in E1, the heterologous sequences can be inserted at or near the site formerly occupied by the deleted E1 sequences, and/or at any other region of the genome. Adenovirus genomes that are mutant in their E1 region can also contain mutations in other regions of the genome, such as the E3 region or the region between E4 and the right end of the genome.
In one embodiment, host cells comprise E1 sequences from an adenovirus of a different type or a different species than the adenovirus vector that is propagated in the host cells. In a preferred embodiment, the host cells comprise human adenovirus E1 sequences and the vector that is propagated in the host cells is a bovine adenovirus.
In one embodiment, the bovine host cells are derived from fetal bovine retina. In a preferred embodiment, fetal bovine retina cells comprise adenovirus E1 sequences that have been introduced into the cells by transfection. In a more preferred embodiment, the E1 sequences are derived from a human adenovirus, for example, human adenovirus type 5 (HAd-5). In a still more preferred embodiment, fetal bovine retina cells, comprising HAd-5 sequences are used for the propagation of replication-defective bovine adenovirus (BAV) vectors having one or more mutations in their E1 region and, optionally, one or more mutations in other regions of their genome. In an even more preferred embodiment, the replication-defective BAV vectors comprise heterologous sequences, wherein the heterologous sequences can be located in the E1 region of the genome of the defective BAV vector and/or at other regions of the genome.
The invention provides host cells as described above, as well as host cells comprising defective BAV vectors mutated in their E1 region, wherein the defective BAV vectors optionally comprise inserted heterologous sequences.
In addition, the invention provides methods for the propagation of replication-defective recombinant BAV vectors using the host cells of the invention, as well as vectors and vector genomes that have been propagated using the host cells of the invention. Defective recombinant BAV vectors and their genomes, produced using the methods and compositions of the invention, are useful as immunogenic compositions. Such immunogenic compositions can be used both prophylactically and therapeutically. Prophylactically, the immunogenic compositions are used for purposes of vaccination to elicit a protective immune response. In their therapeutic uses, the immunogenic compositions are used to induce or boost an immune response to an infection, thereby preventing or ameliorating the symptoms of disease.
In addition, defective recombinant BAV vectors and their genomes, produced using the methods and compositions of the invention, are useful for the introduction of heterologous genes into recipient mammalian cells. When such heterologous genes are in operative linkage with appropriate transcriptional regulatory elements, expression of the heterologous gene in the recipient cell is accomplished. Such expression is useful in the provision of therapeutic gene(s) and/or gene product(s) and thus will play a role in certain aspects of in vitro, in vivo and ex vivo genetic intervention in the treatment of disease, and in gene therapy.